Methods of modulating T cell-mediated immune responses with anti-P-selectin glycoprotein ligand 1 antibodies

ABSTRACT

Compounds that bind to P-Selectin Glycoprotein 1 (PSGL-1) on the surface of T cells or natural killer (NK) cells can be used to induce T cell or NK cell depletion and/or to induce T cell or NK cell apoptosis. The compounds and methods of the invention can be used to control unwanted T cell- or NK cell-mediated immune responses in conditions such as autoimmune diseases, transplant rejection, and allergic diseases.

RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.10/051,497, now U.S. Pat. No. 7,744,888, filed on Jan. 18, 2002, whichclaims priority from U.S. provisional application Ser. No. 60/310,196,filed on Aug. 3, 2001, the entire contents of each of which areincorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to compositions and methods for controlling immuneresponses.

BACKGROUND OF THE INVENTION

The control of unwanted immune responses is a critical issue in thetreatment of diseases such as autoimmune diseases, transplant rejection,allergic diseases, and some cancers. The activity of overly aggressive Tcells can be controlled by immunosuppression or by the induction ofimmunological tolerance. Tolerance is defined as a state where theimmune system is made unresponsive to an antigen, whereasimmunosuppression, which decreases the immune response to antigens,usually requires the continued use of medication. In organtransplantation, T cells play an essential role in the immune responseto alloantigens. Current immunosuppressive regimes commonly involve theuse of corticosteroid, cyclosporin or rapamycin, which block thetranscription of IL-2, a key growth factor for T cells, or inhibit IL-2dependent proliferation. However, a number of monoclonal antibodieswhich either act as T cell-depleting agents (e.g. CD3, CD4, CD8), or asinhibitors of the cytokine signaling or the co-stimulatory pathways of Tcells (e.g. CD25, B7-1, B7-2, CD152, CTLA4) have demonstratedeffectiveness in reducing the incidence of rejection with limited sideeffects or toxicity. Some of these agents have been shown to have somedegree of success in treating autoimmune disease and in prolonging graftsurvival.

Apoptosis is widely believed to be of vital importance for maintainingthe proper function of the immune system and a major mechanism to removeunwanted cells (Kabelitz et al. Immunol. Today 14:338-340 (1993); Raff,Nature:356:397-399 (1992)). Various signals originating from eitherinside or outside a cell influence the life and death of the cell.Antibodies against T cell surface molecules such as Fas (or CD95, MW=43kD), TNFR2 (MW=75 kD), CD2 (MW=45 kD) and CTLA-4 (MW=33 kD) to inducethe apoptosis of T cells (Osborne, Curr. Opin. Immunol. 8:245-248(1996); Lin et al. J. Immunol. 158:598-603 (1997); Zhang et al.Nature:377:348-350 (1995); Lai et al. Eur. J. Immunol. 25:3243-3248(1995); Mollereau et al. J. Immunol. 156:3184-3190 (1996); Gribben etal. Proc. Natl. Acad. Sci. USA 92:811-815 (1995)). Attempts to use Fasand TNFR2 molecules to control unwanted T cells have been hampered bythe fact that these two molecules are expressed not only on immunecells, but also on several other important organ systems like liver.This expression pattern potentially limits the therapeutic applicationof these two antibodies (Ogasawara et al. Nature 364:806-809 (1993);Pfeffer et al. Cell:73:457-467 (1993); Engelmann et al. J. BiologicalChemistry 265:14497-14504 (1990)).

SUMMARY OF THE INVENTION

The invention is based on the discovery that T cells can be depletedand/or induced to undergo apoptosis by the engagement of the T cellsurface antigen P-Selectin Glycoprotein Ligand-1 (PSGL-1). T celldepletion can be particularly useful for the treatment of conditionsassociated with an excessive or unwanted T cell-mediated immune responseor excessive or unwanted T cell proliferation. For example, thedepletion of T cells can cause the reduction or elimination ofundesirable T cell activity or proliferation associated with autoimmunediseases, transplant rejection, allergic diseases, and/or T cell-derivedcancers. The invention encompasses methods of using modulators of PSGL-1function to prevent or reduce a T cell-mediated immune response as wellas methods of screening for modulators of PSGL-1 function.

In one aspect, the invention features a method of preventing or reducinga T cell-mediated immune response in an individual. The method includesthe following steps: selecting an individual diagnosed as having or asbeing at risk of acquiring a condition characterized by an excessive orunwanted T cell-mediated immune response; and administering to theindividual a compound that binds to PSGL-1 on the surface of a T cell,wherein the binding of the compound to PSGL-1 on the surface of the Tcell induces a signal transduction pathway that results in the death ofthe T cell, thereby preventing or reducing a T cell-mediated immuneresponse in the individual.

The compound used in such a method can include an antibody or antigenbinding fragment thereof that specifically binds to PSGL-1. In oneexample, the compound is a monoclonal antibody that specifically bindsto PSGL-1. In one embodiment, the method includes an additional step ofadministering an agent that binds to the monoclonal antibody and inducesthe cross-linking of a plurality of PSGL-1 antigens on the surface ofthe T cell.

In one embodiment, the method includes inducing the cross-linking of aplurality of PSGL-1 antigens on the surface of the T cell, wherein thecross-linking induces the signal transduction pathway that results inthe death of the T cell.

In one example, the method includes a step of selecting an individualdiagnosed as having an autoimmune disease. In another example, themethod includes a step of selecting an individual that has received oris expected to receive an allogeneic or xenogeneic transplant. Inanother example, the method includes a step of selecting an individualdiagnosed as having an allergic disease. In another example, the methodincludes a step of selecting an individual diagnosed as having a T cellcancer.

In one embodiment, the T cell is an activated T cell. In one example,the T cell is a CD4⁺ T cell. In another example, the T cell is a CD8⁺ Tcell.

In one embodiment, the method includes a step of detecting the number ofT cells in a first biological sample taken from the individual beforethe administration of the compound and comparing the results with thenumber of T cells in a second biological sample taken from theindividual after the administration of the compound.

In another embodiment, the method includes a step of detecting abiological activity of T cells in a first biological sample taken fromthe individual before the administration of the compound and comparingthe results with the biological activity of T cells in a secondbiological sample taken from the individual after the administration ofthe compound.

In one embodiment, the administration results in the depletion of atleast 20% of peripheral blood CD3⁺ cells in the individual. In someembodiments, the administration results in the depletion of at least30%, 40%, 50%, or more of the peripheral blood CD3⁺ cells in theindividual.

In one embodiment, the antibody or antigen binding fragment thereofinduces the death of at least 20% of peripheral blood CD3⁺ cells in theindividual after exposure to the antibody or antigen binding fragmentthereof. In some embodiments, the administration induces the death of atleast 30%, 40%, 50%, or more of the peripheral blood CD3⁺ cells in theindividual. Cell death can be measured at any time, e.g., one, two,three, four, five, six, seven, or more days after exposure to theantibody or antigen binding fragment thereof.

In another aspect, the invention features a method of inducing the deathof a T cell or a natural killer (NK) cell. The method includes the stepsof: providing a T cell or NK cell expressing PSGL-1 on its cell surface;and contacting the T cell or NK cell with a compound that binds toPSGL-1 on the surface of the T cell or NK cell, wherein the binding ofthe compound to PSGL-1 on the surface of the T cell or NK cell induces asignal transduction pathway that results in the death of the T cell orNK cell.

The compound used in such a method can include an antibody or antigenbinding fragment thereof that specifically binds to PSGL-1. In oneexample, the compound is a monoclonal antibody that specifically bindsto PSGL-1. In one embodiment, the method includes a step of contactingthe monoclonal antibody with an agent that binds to the monoclonalantibody and induces the cross-linking of a plurality of PSGL-1 antigenson the surface of the T cell or NK cell.

In one embodiment, the method includes a step of inducing thecross-linking of a plurality of PSGL-1 antigens on the surface of the Tcell or NK cell, wherein the cross-linking induces the signaltransduction pathway that results in the death of the T cell or NK cell.

In one embodiment, the T cell is an activated T cell. In one example,the T cell is a CD4⁺ T cell. In another example, the T cell is a CD8⁺ Tcell.

In one embodiment, the method includes a step of assessing the viabilityof the T cell or NK cell after the contacting with the compound.

In one embodiment, the method includes a step of assessing a biologicalactivity of the T cell or NK cell after the contacting with thecompound.

In another aspect, the invention features a method of screening for amodulator of PSGL-1 function. The method includes the steps of:providing a cell expressing PSGL-1 on the surface of the cell;contacting the cell with a test substance; and measuring the viabilityof the cell after contacting the cell with the test substance to therebydetermine if the test substance is a modulator of PSGL-1 function.

In one embodiment, the method includes the step of detecting the deathof the cell induced by the test substance to thereby determine that thetest substance is a modulator of PSGL-1 function.

In one embodiment, the test substance is an antibody or antigen bindingfragment thereof that specifically binds to PSGL-1. In one example, thetest substance is a monoclonal antibody that specifically binds toPSGL-1. In one embodiment, the method includes the step of contactingthe monoclonal antibody with an agent that binds to the monoclonalantibody and induces the cross-linking of a plurality of PSGL-1 antigenson the surface of the cell.

In one embodiment, the method includes the step of inducing thecross-linking of a plurality of PSGL-1 antigens on the surface of thecell, wherein the cross-linking induces the signal transduction pathwaythat results in the death of the cell.

In one embodiment, the T cell is an activated T cell. In one example,the T cell is a CD4⁺ T cell. In another example, the T cell is a CD8⁺ Tcell.

In one embodiment, the method includes the step of manufacturing bulkquantities of the test substance and formulating the test substance in apharmaceutically acceptable carrier.

In another aspect, the invention features a kit containing: a compoundthat binds to PSGL-1 on the surface of a T cell, wherein the binding ofthe compound to PSGL-1 on the surface of the T cell induces a signaltransduction pathway that results in the death of the T cell; andinstructions for use of the compound to treat autoimmunity, transplantrejection, an allergic condition, or a T cell cancer. In otherembodiments, the kit includes instructions for use to treat any diseaseor disorder described herein.

An advantage of the invention is that it allows for the depletion of Tcells and/or the induction of apoptosis in T cells without causing anassociated unwanted or harmful immune response. For example, theadministration of an anti-PSGL-1 antibody to an individual does notresult in an unwanted elevation in the levels of inflammatory cytokinessuch as IL-2 or TNF-α.

Another advantage of the invention is that it allows for the targetingof a cell surface protein, PSGL-1, whose expression is largely limitedto leukocytes, and in particular T cells and NK cells. Therefore, thecompounds described herein do not induce significant levels of apoptosisof other cell types such as liver cells. The targeting of T cells and NKcells (an important CD3⁻ cell type involved in transplantationrejection) for selective depletion, without significantly inducinglife-threatening systemic cytokine responses and damaging other organsystems, is a desired characteristic of an immunosuppressive agent.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the invention, suitable methods and materialsare described below. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In case of a conflict in terminology, the presentspecification will control. In addition, the described materials andmethods are illustrative only and are not intended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the results of a time-course experiment that investigatedwhen activated T cells acquire sensitivity to TAB4 (an anti-PSGL-1monoclonal antibody)-mediated apoptotic signals.

FIG. 2 depicts the results of cell surface biotinylation andimmunoprecipitation of the antigen recognized by the TAB4 antibody.

FIG. 3 depicts the expression of the PSGL-1 antigen on spleen CD4⁺ Tcells, CD8⁺ T cells, CD19⁺ B cells, and NK cells.

FIG. 4 depicts the expression of the PSGL-1 antigen on CD4⁺, CD8⁺, andCD4⁺8⁺, and CD4⁻8⁻ thymocytes.

FIG. 5 depicts the levels of IL-2 produced in mixed lymphocyte cultureusing spleen cells isolated from TAB4 (or hamster Ig)-treated BALB/cmice as the responders and H2-mismatched C3H spleen cells as thestimulator.

FIG. 6 depicts western blot analyses demonstrating that (A) proteinsimmunoprecipitated with the TAB4 antibody can be recognized by acommercially available anti-PSGL-1 antibody and (B) preclearing of Tcell lysate with anti-PSGL-1 antibody can deplete the proteinsrecognized by the TAB4.

FIG. 7 depicts the percentage of surviving grafts in C57BL/6 mice thatreceived a skin graft from BALB/c mice and were treated with ananti-PSGL-1 antibody (closed diamond) or a control antibody (opensquare).

FIG. 8 depicts the time course of the percentage of apoptotic T cellsfollowing the treatment of activated human peripheral blood mononuclearcells with an anti-human PSGL-1 antibody.

FIG. 9 depicts the incidence of diabetes in autoimmune non-obesediabetic (NOD) male mice that were treated with anti-PSGL-1 antibody(closed square) or a control antibody (open square).

DETAILED DESCRIPTION

The invention is directed to methods of modulating T cell activity bymodulating the function of PSGL-1 molecules residing on the surface of aT cell. Engagement of PSGL-1 with compositions described herein cancause the depletion of T cells and/or induce T cells to undergoapoptosis. These compositions are therefore useful as therapeutic agentsfor controlling immune-related conditions such as autoimmune diseases,transplant rejection, allergic diseases, and/or T cell-derived cancers.The compositions are also useful in causing the depletion of T cellsfrom any biological sample where the presence or activity of T cells isnot desired.

PSGL-1 Protein

PSGL-1 is a cell surface adhesion molecule that is expressed onneutrophils, T and B-lymphocytes, NK cells, monocytes, dendritic cells,and primitive human CD34 hematopoietic progenitor cells. Through itsability to interact with selectins, PSGL-1 mediates the rolling ofleukocytes on the endothelium and the extravasation of leukocytes intoinflamed tissues. PSGL-1-mediated binding of T cells to E- andP-selectin, or migration, is differentially regulated. For instance, theappearance of CLA (cutaneous lymphocyte antigen) epitope is thought tobe induced on T cells undergoing naive to memory transition. Onlyactivated helper 1 but not helper 2 T cells express functional PSGL-1and it capable of migration into the inflamed area of the skin.

PSGL-1 is a sialomucin that must be specifically sialylated,fucosylated, and sulfated to bind P-selectin. The PSGL-1 molecule existsin isoforms characterized by different degree of glycosylation andsulfation sites at their N-termini. Resting peripheral blood T and Bcells, lymphoid cell lines, and in vitro activated peripheral blood Tcells express similar level of PGSL-1. Yet, only activated T cellsdisplay a functional form of PSGL-1 and bind avidly to P-selectin. Suchactivation-dependent binding activity appears to be a result ofdifferential post-translational modification, as suggested by elevatedlevels of alpha (1,3) fucosyltransferases activities in activated Tcells. PSGL-1 isoforms also show differential affinity to L-selectin andE-selectin. For instance, human T cells exhibiting the CLA-positiveisoform can tether and roll on both E- and P-selectin, while T cellsexpressing PSGL-1 without the CLA epitope only bind to P-selectin.Furthermore, binding of PSGL-1 to P-selectin is contingent upon thepresence of the terminal decapeptide that contains three tyrosineresidues for sulfation and one threonine residue for glycosylation.

A PSGL-1 protein can be prepared by recombinant methods and/or byisolating a native PSGL-1 protein from biological material. Arecombinant PSGL-1 protein can be produced in prokaryotic or eukaryoticcells, either in vitro or in vivo. Nucleic acids encoding PSGL-1 can beused for recombinant production of the protein (see, e.g., GenBank™Accession NM_(—)003006 for an example of a nucleic acid encoding aPSGL-1 polypeptide). Antibodies directed to PSGL-1 are also well knownand can be used for purification of the antigen (see, e.g., Herron etal. (2000) Science June 2; 288(5471):1653-56; WO 00/25808) and/or usedin methods described herein. PSGL-1 is further described in referencesincluding but not limited to Sako et al. (1993) Cell 75:1179; Vachino etal. (1995) J. Biol. Chem. 270:21966; and Veldman et al. (1995) J. Biol.Chem. 270:16470.

For recombinant production of PSGL-1, the simultaneous expression ofboth PSGL-1 and its modifying alpha-(1,3) fucosyltransferase, Fuc-TVII,may be required for the functional expression of PSGL-1. In addition oralternatively, recombinant production of PSGL-1 may be accompanied byco-transfection with a nucleic acid encoding PACE for removing thepropeptide and/or or a nucleic acid encoding tyrosine sulfotransferase.

An anti-PSGL-1 antibody can be used to isolate and purify a PSGL-1antigen from biological material. Any cell type expressing a PSGL-1protein, e.g., T cells derived from an individual or a T cell line, canbe used as a source of the protein. Once purified, the protein can beused in a variety of methods as described herein. For example, thepurified PSGL-1 protein can be used in an in vitro screen of modulatorsof PSGL-1 function on T cells or as an immunogen to prepare antibodiesdirected against the protein.

In addition, a PSGL-1 antigen can be purified using a selectin-Fcfusion, e.g., a P-selectin-Fc fusion.

Anti-PS GL-1 Antibodies

PSGL-1 polypeptides (or immunogenic fragments or analogs thereof) can beused to generate antibodies useful in the methods of the invention. Asdescribed above, PSGL-1 polypeptides or peptide fragments thereof can beproduced by recombinant techniques or synthesized using solid phasesynthesis methods. The recombinant PSGL-1 polypeptides or a peptidefragment thereof can be used as an immunogen to produce anti-PSGL-1antibodies. In addition, an anti-PSGL-1 antibody, such as the TAB4monoclonal antibody, can be used to purify a PSGL-1 polypeptide, e.g., aPSGL-1 polypeptide in its natural conformation, which can then be usedas an immunogen to produce additional anti-PSGL-1 antibodies.

An antibody of the invention can be a monoclonal, polyclonal, orengineered antibody that specifically binds to a PSGL-1 polypeptide. Anantibody that “specifically binds” to a particular antigen, e.g., aPSGL-1 polypeptide, will not substantially recognize or bind to othermolecules in a sample. Thus, the invention also features methods foridentifying a test compound (e.g., an antibody) that binds to apolypeptide of the invention by contacting the polypeptide with a testcompound and determining whether the polypeptide binds to the testcompound (e.g., by direct detection of the binding, detection of acompetitor molecule which disrupts binding of the test compound to thepolypeptide, and/or detection of binding using an assay forapoptosis-inducing activity).

In general, PSGL-1 polypeptides can be coupled to a carrier protein,such as KLH, mixed with an adjuvant, and injected into a host mammal.Antibodies produced in that animal can then be purified by peptideantigen affinity chromatography.

In particular, various host animals can be immunized by injection with aPSGL-1 polypeptide or an antigenic fragment thereof. Commonly employedhost animals include rabbits, mice, guinea pigs, and rats. Variousadjuvants that can be used to increase the immunological response dependon the host species and include Freund's adjuvant (complete andincomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Potentiallyuseful human adjuvants include BCG (bacille Calmette-Guerin) andCorynebacterium parvum. Polyclonal antibodies are heterogeneouspopulations of antibody molecules that are contained in the sera of theimmunized animals.

Antibodies within the invention therefore include polyclonal antibodiesand, in addition, monoclonal antibodies, humanized or chimericantibodies, single chain antibodies, Fab fragments, F(ab′)₂ fragments,and molecules produced using a Fab expression library.

Monoclonal antibodies, which are homogeneous populations of antibodiesto a particular antigen, can be prepared using the PSGL-1 polypeptidesdescribed above and standard hybridoma technology (see, for example,Kohler et al., Nature 256:495 (1975); Kohler et al., Eur J Immunol 6:511(1976); Kohler et al., Eur J Immunol 6:292 (1976); Hammerling et al.,Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y. (1981)).

In particular, monoclonal antibodies can be obtained by any techniquethat provides for the production of antibody molecules by continuouscell lines in culture such as described in Kohler et al., Nature 256:495(1975), and U.S. Pat. No. 4,376,110; the human B-cell hybridomatechnique (Kosbor et al., Immunology Today 4:72 (1983); Cole et al.,Proc Natl Acad Sci USA 80:2026 (1983)), and the EBV-hybridoma technique(Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96 (1983)). Such antibodies can be of any immunoglobulinclass including IgG, IgM, IgE, IgA, IgD and any subclass thereof. Thehybridoma producing the mAb of this invention may be cultivated in vitroor in vivo. The ability to produce high titers of mAbs in vivo makesthis a particularly useful method of production.

Once produced, polyclonal or monoclonal antibodies are tested forspecific PSGL-1 recognition by Western blot or immunoprecipitationanalysis by standard methods, for example, as described in Ausubel etal., supra. Antibodies that specifically recognize and bind to PSGL-1are useful in the invention. Anti-PSGL-1 antibodies that bind to thePSGL-1 antigen on the surface of a T cell, e.g., a CD3⁺ cell, and inducethe depletion and/or apoptosis of T cells in an individual areparticularly useful.

The antibodies can be used, for example, as part of a therapeuticregimen (e.g., to reduce or eliminate an undesirable immune response,such as a T cell mediated immune response, associated with conditionssuch as autoimmune diseases, transplant rejection, allergic diseases,and T cell-derived cancers). Antibodies also can be used in a screeningassay to measure the ability of a candidate compound to bind to PSGL-1.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc Natl Acad Sci USA 81:6851 (1984);Neuberger et al., Nature 312:604 (1984); Takeda et al., Nature 314:452(1984)) by splicing the genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Achimeric antibody is a molecule in which different portions are derivedfrom different animal species, such as those having a variable regionderived from a murine monoclonal antibody and a human immunoglobulinconstant region.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. Nos. 4,946,778, 4,946,778, and 4,704,692) can beadapted to produce single chain antibodies against a PSGL-1 polypeptide,or a fragment thereof. Single chain antibodies are formed by linking theheavy and light chain fragments of the Fv region via an amino acidbridge, resulting in a single chain polypeptide.

Antibody fragments that recognize and bind to specific epitopes can begenerated by known techniques. For example, such fragments include butare not limited to F(ab′)₂ fragments that can be produced by pepsindigestion of the antibody molecule, and Fab fragments that can begenerated by reducing the disulfide bridges of F(ab′)₂ fragments.Alternatively, Fab expression libraries can be constructed (Huse et al.,Science 246:1275 (1989)) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

Antibodies can be humanized by methods known in the art. For example,monoclonal antibodies with a desired binding specificity can becommercially humanized (Scotgene, Scotland; Oxford Molecular, Palo Alto,Calif.). Fully human antibodies, such as those expressed in transgenicanimals are also features of the invention (Green et al., NatureGenetics 7:13 (1994); and U.S. Pat. Nos. 5,545,806 and 5,569,825).

Screening Assays for Compounds that Modulate PSGL-1 Function

The invention also encompasses methods for identifying compounds thatinteract with PSGL-1 (or a domain of PSGL-1) including, but not limitedto, compounds that induce T cell depletion and/or T cell apoptosis uponbinding to PSGL-1. Also included are compounds that modulate theinteraction of PSGL-1 with transmembrane, extracellular, orintracellular proteins that regulate PSGL-1 activity and compounds whichmodulate PSGL-1 activity.

The compounds that may be screened in accordance with the inventioninclude, but are not limited to peptides, antibodies and fragmentsthereof, and other organic compounds that bind to PSGL-1 and modulate abiological function mediated by PSGL-1, as described herein.

Such compounds may include, but are not limited to, peptides such as,for example, soluble peptides, including but not limited to members ofrandom peptide libraries; (Lam et al., Nature 354:82 (1991); Houghten etal., Nature 354:84 (1991)), and combinatorial chemistry-derivedmolecular library made of D- and/or L configuration amino acids,phosphopeptides (including, but not limited to, members of random orpartially degenerate, directed phosphopeptide libraries; Songyang etal., Cell 72:767 (1993)), antibodies (including, but not limited to,polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or singlechain antibodies, and Fab, F(ab′)₂ and Fab expression library fragments,and epitope-binding fragments thereof), and small organic or inorganicmolecules.

Other compounds which can be screened in accordance with the inventioninclude but are not limited to small organic molecules that affect anactivity of the PSGL-1 protein, as described herein.

Computer modeling and searching technologies permit identification ofcompounds, or the improvement of already identified compounds, that canmodulate PSGL-1 expression or activity. Having identified such acompound or composition, the active sites or regions are identified.Such active sites might typically be a binding site for a naturalmodulator of activity. The active site can be identified using methodsknown in the art including, for example, from the amino acid sequencesof peptides, from the nucleotide sequences of nucleic acids, or fromstudy of complexes of the relevant compound or composition with itsnatural ligand. In the latter case, chemical or X-ray crystallographicmethods can be used to find the active site by finding where on thefactor the modulator (or ligand) is found.

Although described above with reference to design and generation ofcompounds which could alter binding, one could also screen libraries ofknown compounds, including natural products or synthetic chemicals, andbiologically active materials, including proteins, for compounds whichbind to a PSGL-1 protein and cause T cell depletion and/or induce T cellapoptosis.

In vitro systems may be designed to identify compounds capable ofinteracting with PSGL-1 (or a domain of PSGL-1). Compounds identifiedmay be useful, for example, in modulating T cell activity as describedherein and thus may be useful for the treatment of conditions such asautoimmune diseases, transplant rejection, allergic diseases, and Tcell-derived cancers.

The principle of the assays used to identify compounds that bind toPSGL-1 involves preparing a reaction mixture of PSGL-1 (or a domainthereof) and the test compound under conditions and for a timesufficient to allow the two components to interact and bind, thusforming a complex which can be removed and/or detected in the reactionmixture. The PSGL-1 species used can vary depending upon the goal of thescreening assay. In some situations it is preferable to employ a peptidecorresponding to a domain of PSGL-1 fused to a heterologous protein orpolypeptide that affords advantages in the assay system (e.g., labeling,isolation of the resulting complex, etc.) can be utilized.

The screening assays can be conducted in a variety of ways. For example,one method to conduct such an assay involves anchoring PSGL-1 protein,polypeptide, peptide or fusion protein or the test substance onto asolid phase and detecting PSGL-1/test compound complexes anchored on thesolid phase at the end of the reaction. In one embodiment of such amethod, the PSGL-1 reactant may be anchored onto a solid surface, andthe test compound, which is not anchored, may be labeled, eitherdirectly or indirectly.

In practice, microtiter plates may conveniently be utilized as the solidphase. The anchored component may be immobilized by non-covalent orcovalent attachments. Non-covalent attachment may be accomplished bysimply coating the solid surface with a solution of the protein anddrying. Alternatively, an immobilized antibody, preferably a monoclonalantibody, specific for the protein to be immobilized may be used toanchor the protein to the solid surface. The surfaces may be prepared inadvance and stored.

In order to conduct the assay, the nonimmobilized component is added tothe coated surface containing the anchored component. After the reactionis complete, unreacted components are removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized on thesolid surface. The detection of complexes anchored on the solid surfacecan be accomplished in a number of ways. Where the previouslynon-immobilized component is pre-labeled, the detection of labelimmobilized on the surface indicates that complexes were formed. Wherethe previously non-immobilized component is not pre-labeled, an indirectlabel can be used to detect complexes anchored on the surface; e.g.,using a labeled antibody specific for the previously non-immobilizedcomponent (the antibody, in turn, may be directly labeled or indirectlylabeled with a labeled anti-Ig antibody).

Alternatively, a reaction can be conducted in a liquid phase, thereaction products separated from unreacted components, and complexesdetected; e.g., using an immobilized antibody specific for PSGL-1protein, polypeptide, peptide or fusion protein or the test compound toanchor any complexes formed in solution, and a labeled antibody specificfor the other component of the possible complex to detect anchoredcomplexes.

Alternatively, cell-based assays can be used to identify compounds thatinteract with PSGL-1. To this end, cell lines that express PSGL-1, orcell lines that have been genetically engineered to express PSGL-1 canbe used. Cell based assays are particularly useful for evaluating thefunctional effects of a compound identified by a screen describedherein. For example, once a compound is identified based upon itsability to bind to a PSGL-1 protein, the compound can then be tested forits ability to, e.g., induce T cell apoptosis in vitro or in vivo ordeplete T cells in vitro or in vivo.

Pharmaceutical Compositions

Given that an object of the present invention is to alter an immuneresponse in an individual, a pharmaceutical composition containing, forexample, antibodies, small molecules, or other compounds thatspecifically bind PSGL-1 polypeptides are also a feature of theinvention. In a preferred example, the compound functions as an agonistof PSGL-1.

Pharmaceutical compositions for use in accordance with the presentinvention can be formulated in a conventional manner using one or morephysiologically acceptable carriers or excipients. Thus, the compoundsand their physiologically acceptable salts and solvates may beformulated for administration by a variety of routes of administration.

The compounds may be formulated for parenteral administration byinjection, for example, by bolus injection or continuous infusion.Formulations for injection may be presented in unit dosage form, forexample, in ampoules or in multi-dose containers, with an addedpreservative. The compositions may take such forms as suspensions,solutions, or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient can be in powder form forconstitution with a suitable vehicle, for example, sterile pyrogen-freewater, before use.

Methods of Controlling a T Cell-Mediated Immune Response and Depleting TCell Populations

Compounds such as those detailed in the screening assays describedherein may be useful, for example, in modulating a biological functionmediated by a PSGL-1 polypeptide and/or for the treatment of disordersassociated an excessive or unwanted immune response, e.g., a Tcell-mediated immune response. These compounds include, but are notlimited to peptides, antibodies and fragments thereof, and other organiccompounds that bind to PSGL-1 on the surface of a T cell and induce asignal transduction pathway that results in the death of the T cell. Themethods of the invention optionally include the addition of across-linking agent that induces the cross-linking of PSGL-1 on thesurface of a cell. The compounds described herein can be used in anyinstance wherein the depletion or elimination of T cell activity isdesired. Particularly useful conditions that can be treated with thecompounds of the invention include autoimmune diseases, transplantrejection, allergic diseases, and T cell-derived cancers.

Examples of conditions that can be treated with the anti-PSGL-1compounds described herein include, but are not limited to, diabetesmellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoidarthritis, osteoarthritis, and psoriatic arthritis), multiple sclerosis,encephalomyelitis, myasthenia gravis, systemic lupus erythematosus,autoimmune thyroiditis, dermatitis (including atopic dermatitis andeczematous dermatitis), psoriasis, Sjogren's Syndrome, Crohn's disease,aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, type Idiabetes, inflammatory bowel diseases, ulcerative colitis, asthma,allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis,proctitis, drug eruptions, leprosy reversal reactions, erythema nodosumleprosum, autoimmune uveitis, allergic encephalomyelitis, acutenecrotizing hemorrhagic encephalopathy, idiopathic bilateral progressivesensorineural hearing loss, aplastic anemia, pure red cell anemia,idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis,chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue,lichen planus, Graves' disease, sarcoidosis, primary biliary cirrhosis,uveitis posterior, interstitial lung fibrosis, graft-versus-hostdisease, cases of transplantation (including transplantation usingallogeneic or xenogeneic tissues) such as bone marrow transplantation,liver transplantation, or the transplantation of any organ or tissue,allergies such as atopic allergy, AIDS, and T-cell neoplasms such asleukemias and/or lymphomas.

The methods of the invention can be used to deplete T cells from a cellpopulation, either in vitro or in vivo. For example, a biological samplederived from an individual can be depleted of T cells in vitro bycontacting the sample with an anti-PSGL-1 compound described herein,optionally together with a cross-linking agent. This method can beuseful, e.g., by allowing for the enrichment of non-T cells in a cellpopulation as well as by reducing or eliminating T cell activity from acell population.

The following are examples of the practice of the invention. They arenot to be construed as limiting the scope of the invention in any way.

EXAMPLES Example 1 Preparation of an Anti-T Cell Apoptosis InducingProtein (“TAIP”) Monoclonal Antibody

A TAIP-specific monoclonal antibody was generated by applying the wellknown cell fusion methods of Kohler and Milstein ((1976) EuropeanJournal of Immunology 6:511-519) to produce a hybridoma secretingdesired antibodies. Antibody-producing cells from a hamster injectedwith Concanavalin A (Con A)-activated BALB/c spleen T cells were fusedwith a myeloma cell line to form an antibody secreting hybridoma. Thetwo populations of cells were fused with polyethylene glycol, and theresulting antibody producing cells were cloned and propagated bystandard tissue culture methods. One hybridoma generated according tothese methods secreted a monoclonal antibody, designated TAB4, that wasable to induce T cell apoptosis in vitro and deplete T cells in vivo.The protein recognized by TAB4 was designated T cell apoptosis inducingprotein (TAIP).

C57BL/6J (B6) and BALB/c mice were purchased from the Jackson lab (BarHarbor, Me.). Syrian hamsters were purchased from the Animal CoreFacility, National Taiwan University Medical College.

Concentrated culture supernatant of the TAB4 hybridoma was spun at20,000×g for 10 minutes and the supernatant was diluted at a 1:1 ratiowith the binding buffer (0.1 M sodium acetate, pH 5.0). A protein Gcolumn (approximately 1 ml bed volume) was washed three times with 3-5ml of the binding buffer. The cleared culture supernatant was loaded tothe protein G column and the flow-through was collected and reloaded tothe column. The column was washed with 6-10 ml of the binding buffer andthe bound antibody was eluted from the column with 5 ml of the elutionbuffer (0.1 M glycine-HCl, pH 2.8). Each fraction contained 1 ml of theeluted antibody and the eluted fraction was adjusted to neutral pH bymixing each 1 ml fraction with 50 microliters of 1 M Tris-HCl, pH 7.5.Fractions containing the antibody were pooled and dialyzed against 2liters of PBS, pH 7.4 three times at three hours for each dialysis.Protein concentration in the antibody samples were determined with theprocedure described by Bradford using the Bio-Rad Protein Assay(BIO-RAD, Hercules, Calif.).

Example 2 Preparation of a Mouse Spleen Cell Suspension and theActivation and Enrichment of T Cells

Mouse spleen was immersed in 8 ml of Hank's balanced salt solution(HBSS), gently minced with a sterile cover slip, transferred to a 15 mlcentrifuge tube (Costar), and spun at 200×g for 5 minutes. Thesupernatant was discarded and the cell pellet was resuspended in theresidual buffer by gently tapping the wall. The contaminating red bloodcells (RBC) were lysed by the addition of 1 ml of RBC lysis buffer (0.6M NH₄Cl, 0.17 M Tris-base, pH 7.65), followed by a 2 min incubation atroom temperature and rapid quenching with 9 ml of HBSS. The cells werepelleted at 200×g for 5 minutes, washed twice and resuspended in RPMImedium. The concentration and viability of cells in the mixture weredetermined with a hemocytometer (Cambridge Scientific Inc.) and Trypanblue exclusion.

The spleen cells were adjusted to a final concentration of 3×10⁶/ml withRPMI medium and Concanavalin A was added to a final concentration of 2micrograms/ml to activate the T cells. The cell suspension wastransferred to a 6-well culture plate (5 ml/well) or a 10-cm culturedish (10 ml/dish) and incubated at 37° C., 5% CO₂ for 48 hours beforeharvesting. The activated spleen cells, including activated T cells,were resuspended in 5 ml of HBSS and carefully overlaid on top of a 5 ml55% cushion of Percoll solution in a centrifuge tube. Care was taken notto disturb the separated layers. The cells were spun at 1,900×g for 13minutes at 25° C. without the brake. The enriched T cells were collectedfrom the interface of the two layers, washed twice with HBSS, and wereready for experiments.

Example 3 Apoptosis of Activated T cells

Activated T cells (see Example 2) were resuspended to a finalconcentration of 5×10⁵ cells/ml in RPMI medium containing 5 ng/ml ofIL-2, and treated with control Ig, TAB4, or anti-CD3 according to theconditions shown in Table 1.

After an incubation period of 18-24 hours, the extent of apoptosis ineach culture was determined using the 7-AAD apoptosis assay. The treatedcells were transferred to FACS tubes (Falcon), washed twice withice-cold FACS solution (1% fetal bovine serum, 0.05% sodium azide inPBS), pelleted at 200×g at 4° C. The cells were resuspended in ice-coldFACS solution to a final concentration of 1-2×10⁷ cells/ml. Forstaining, 0.1 ml of the resuspended cells were mixed with 7-AAD to afinal concentration of 2 ug/ml and then incubated at 4° C. in the darkfor 20 minutes. Finally, the stained cells were washed twice withice-cold FACS solution, resuspended in 0.5 ml of FACS solution andanalyzed with BD LSR flow cytometer (Beckton Dickinson).

TABLE 1 Experiment groups Treatment* Negative control 3 ug/ml hamster Ig5 ng/ml IL-2 3 ug/ml cross-linker antibody (anti-hamster Ig) TAB4 3ug/ml TAB4 hamster mAb 5 ng/ml IL-2 3 ug/ml cross-linker antibody(anti-hamster Ig) Positive control 1 ug/ml anti-CD3 mAb 5 ng/ml IL-2 1ug/ml cross-linker antibody (anti-mouse Ig) *Final concentration of thedesignated reagents in the medium.

FIG. 1 depicts the results of a representative time-course experimentthat investigated when activated T cells acquire sensitivity to TAB4(anti-TAIP)-mediated apoptotic signals. Mouse splenocytes were activatedwith Con-A and maintained in IL-2 containing medium. Activated T cellswere harvested, resuspended, and challenged with TAB4 monoclonalantibody or control hamster IgG in the presence of anti-hamster IgGantibody as cross-linker. The ability of TAIP cross-linking to inducelow level (6.5%) of apoptotic cell death was evident on day one.However, the extent of TAB4-induced apoptosis increased from 17% on day2, peaked at 52% on day 4, and declined to 44% on day 6. The controlhamster IgG did not induce specific apoptotic T cell death, as comparedwith the cultures that received only IL-2. Anti-CD3 (as positivecontrol) induced apoptosis in 38% of T cell after 48 hours of activation(data not shown).

Example 4 Expression of the TAIP Antigen in Different Tissues

Cells were washed twice with ice-cold FACS solution (1% fetal bovineserum, 0.05% sodium azide in PBS) and spun at 200×g at 4° C. in a FACStube (FALCON™). The cells were resuspended in ice-cold FACS solution toa final concentration of 1×10⁷ cells/ml and a 0.1 ml aliquot of theresuspended cells in a FACS tube (FALCON™) was used for each assay. Forsurface staining, the TAB4 monoclonal antibody or a control hamster Igat a final concentration of 2 ug/ml were added to the cells and themixtures were incubated at 4° C. for 30 minutes in the dark. The cellswere washed once with ice-cold FACS and then stained with: (1) forspleen cells, cychrome-conjugated anti-CD3 antibody (2 ug/ml),FITC-conjugated anti-hamster Ig, and PE-conjugatedanti-CD8/CD4/CD19/CD11b/pan-NK/I-A/I-E- /Mac-3 antibody (2 ug/ml) in 100ul of ice-cold FACS solution; and (2) for thymus cells, FITC-conjugatedanti-hamster Ig, PE-conjugated anti-CD8, and cychrome-conjugatedanti-CD4 antibodies (2 ug/ml) in 100 ul of ice-cold FACS solution. Thereaction was performed at 4° C. for 30 minutes in the dark. Finally, thestained cells were washed twice with ice-cold FACS solution, resuspendedin 1 ml of FACS solution and analyzed with BD™ LSR flow cytometer(Beckton Dickinson).

FIGS. 3 and 4 demonstrate by FACS analysis the distribution of TAIPantigen on the various subpopulations of splenocytes and thymocytes. Asshown in FIG. 3, CD19⁺ B cells expressed low but detectable amounts ofTAIP proteins on the surface. Significantly higher amounts of TAIPproteins were detected on CD3⁺ T cells and a fraction of NK cells. Mostof the CD4⁺, CD8⁺, and CD4⁺8⁺ thymus T cells expressed significantamounts of TAIP proteins. In contrast, the TAIP proteins were expressedonly on a small population of CD4⁻8⁻ thymus T cells (FIG. 4).

Tissues from B6 and BALB/c mice, including brain, thymus, heart, lung,liver, stomach, kidney, spleen, and skin, were collected, fixed in 10%formaldehyde overnight at room temperature, and embedded in paraffinblocks. Tissue sections, at a 4 um thickness, were prepared from theparaffin block with Leica RM2135 microtome, spread in 45° C. water, andlaid on a coated slide. The slides were dried in 37° C. and were readyfor subsequent experiments.

Slides containing the tissue paraffin sections were dewaxed and driedthrough a xylenes-100% ethanol series according to standard protocol andwere finally kept in 100% ethanol. The sections were rehydrated througha 100% ethanol-90% ethanol-85% ethanol-70% ethanol-PBS serial incubationaccording to standard protocol to a final PBS solution. The followingreactions were all performed in a humidified box. Non-specific bindingwere blocked by incubating the tissue sections in blocking buffer (1%normal goat serum) for 1 hour at room temperature (or 4° C. overnight).The blocking buffer was removed and TAB4 or normal hamster Ig (1:200dilution) was added to the sections and incubation continued for anotherhour at room temperature (or 4° C. overnight). The sections were washedtwice in PBS, for 5 minutes each, to remove the primary antibody,reacted with 1:250 diluted alkaline phosphatase-conjugated goatanti-hamster Ig, and incubated at room temperature for 1 hour. Thesections were again washed twice with PBS, 5 minutes each, to remove theantibody-enzyme conjugate and the color reaction was developed withBCIP/NBT substrate solution at room temperature for 30 minutes in thedark. The sections were washed again with PBS to remove excess enzymesubstrate, dehydrated through the PBS-ethanol-xylenes series, andmounted for microscopy.

The results indicated that the TAIP proteins expression were detectedonly in bone marrow derived-tissues but not on the rest of the tissuestested.

Example 5 Cell Surface Biotinylation and Immunoprecipitation of the TAIPAntigen

5×10⁷ RL♂1 or NIH-3 T3 cells were surface biotinylated in 1 ml of PBScontaining 0.5 mg/ml Sulfo-NHS-biotin (Pierce) for 30 minutes on ice.The reaction was terminated by incubating the cells with 0.5 ml ofDulbecco's modified Eagle's medium (Life Technologies, Inc.) for 10minutes on ice. Cells were washed with 1 ml of Dulbecco's modifiedEagle's medium once and with 1 ml of phosphate-buffered saline twice.

Labeled cells were lysed at a density of 5.0×10⁷ cells/ml in cold lysisbuffer (1% Triton X-100, 20 mM Tris-HCl, pH 8.0, 160 mM NaCl, 1 mMCaCl₂) containing complete protease inhibitor cocktail (Roche) for 15minutes, and insoluble material was pelleted at 10,000×g for 10 minutes;these and all subsequent steps were performed at 4° C. Forimmunoprecipitation, the lysate was preincubated for 30 minutes with 50μl of packed protein G-Sepharose (Amersham Pharmacia Biotech) to removenon-specifically binding proteins. Beads were pelleted, and aliquots ofthe supernatant (routinely corresponding to 5.0×10⁷ cells) wereincubated with 20 μl of protein G-Sepharose preloaded with 10 μg of mAbTAB4 or IgG from normal hamster serum. After incubation for 4 h at 4°C., the resin was washed four times with washing buffer (0.05% TritonX-100, 50 mM Tris-HCl, pH 8.5, 400 mM NaCl, 1 mM CaCl₂, 1 mg/mlovalbumin), twice with a similar washing buffer, containing 250 mMinstead of 400 mM NaCl. Proteins specifically bound to the TAB4 wereeluted with 50 μl of 1×SDS sample buffer. Eluted proteins were separatedby 8% SDS-PAGE and transferred to nitrocellulose membrane (Millipore).Filters were analyzed for biotinylated proteins withperoxidase-conjugated Avidin (PHARMINGEN™) and developed with theChemiluminescence reagent (NEN™ Life Science Products).

As shown in FIG. 2, a biotinylated surface protein with a molecularweight of approximately 120-kD was identified by TAB4 in RL♂1 cells(TAIP⁺ T cells), but not in 3T3 cells (TAIP⁻ cells). In contrast,protein G sepharose coated with hamster normal serum could not retrievethis 120-kDa protein. These results suggest that this 120-kDa protein isthe antigen recognized by monoclonal antibody TAB4 on the cell surfaceof T cells.

Example 6 Depletion of T Cells In Vivo

To examine the effects of TAB4 on populations of T cells and other cellsin vivo, mice were injected with 300 ug of TAB4 or control hamster Igintraperitoneally and, on day 4, splenocytes, thymocytes, and peripheralblood mononuclear cells were harvested for the total cell count and forthe analyses of cell surface markers by FACS.

For FACS assays, the cells were fixed with 2% paraformaldehyde at 4° C.for 20 minutes, washed twice, and resuspended in ice-cold FACS solutionto a final concentration of 1×10⁷ cells/ml. A 100 μl aliquot of theresuspended cells in a FACS tube (FALCON™) was used for each assay. TAB4or control hamster Ig at a final concentration of 2 ug/ml were added tothe cells and the mixtures were incubated at 4° C. for 30 minutes in thedark. The cells were washed once with ice-cold FACS and reacted with:(1) for spleen cells, cychrome-conjugated anti-CD3 antibody (2 ug/ml),FITC-conjugated anti-hamster Ig and PE-conjugatedanti-CD8/CD4/CD19/CD11b/pan-NK/I-A/I-E/Mac-3 antibody (2 ug/ml) in 100ul of ice-cold FACS solution; and (2) for thymus cells, FITC-conjugatedanti-hamster Ig, PE-conjugated anti-CD8, and cychrome-conjugatedanti-CD4 antibodies (2 ug/ml) in 100 ul of ice-cold FACS solution. Thereaction was performed at 4° C. for 30 minutes in the dark. Finally, thestained cells were washed twice with ice-cold FACS solution, resuspendedin 1,000 ul of FACS solution and analyzed with BD™ LSR flow cytometer(Beckton Dickinson).

Four days after the injection, the percentages of CD3⁺ T cells inperipheral blood leukocytes (PBL) decreased from 36.7% in control miceto 4.1% in TAB4-treated mice (Table 2). TAB4 treatment caused a slightreduction in the total number of splenocytes. However, in TAB4 treatedmice, there was a 62% decrease in the number of CD3⁺ T cells, a 50%decrease in the number of NK cells, and a slight increase in the totalnumber of CD19⁺ B cells. The total number of thymocytes recovered fromTAB4 treated mice was only 48% of the level seen in control (52%reduced). Moreover, except for CD4⁺ T cells, all other CD8⁺, CD4⁺CD8⁺,and CD4⁻CD8⁻ T cells were reduced, with CD4⁺CD8⁺ subpopulation being themost profoundly affected (64.7% reduction).

TABLE 2 No Normal TAB4- Depletion ×10⁶ Treatment Hamster Ig treated (%)Spleen Total 123 93.3 105 14.6 Splenocytes CD3⁺ T cells 32.8 28.4 12.462.2 CD3⁻ CD19⁺ 72.2 53.4 72.9 −0.8 CD3⁻ NK⁺ 3.6 2.4 1.80 50 PeripheralBlood Leukocytes CD3⁺ T cells 36.7% 36% 4.1% 88.8% Thymus Total 94 22945 52.1 Thymocytes CD4⁺ 9.3 28.4 10.9 −16.6 CD8⁺ 5.2 7.7 3.6 30.3 CD4⁺CD8⁺ 73.8 182 26 64.7 CD4⁻ CD8⁻ 5.6 10.5 4.5 19.3 (representative datafrom three experiments)

Example 7 Anti-TAIP Antibody does not Induce IL-2 or TNF-Alpha Secretion

BALB/c mice (H-2d) were intraperitoneally injected with 300 microgramsof TAB4 or control hamster Ig. Splenocytes were isolated 7 days afterinjection, and used as responders in culture with mitomycin C-treatedC3H(H-2k) splenocytes (as stimulators). Three days later, the culturesupernatants were harvested and the IL-2 content was measured by ELISAset ( PHARMINGEN™). As shown in FIG. 5, the IL-2 production wassuppressed in responder cells derived from TAB4-treated mice as comparedwith that of control mice. The plasma levels of IL-2 and TNF-alpha werealso analyzed and no significant difference was noted in the levels ofIL-2 (or TNF-alpha) in the sera of the control and the TAB4 treatedmice. Since production of IL-2 is central to the activity of T cells,the results show that a TAIP-specific antibody, such as TAB4, can beused in vivo to manipulate T cells and control unwanted T cell-mediatedimmune responses such as those associated with autoimmune diseases andtransplantation rejection.

Example 8 Use of an Anti-TAIP Antibody to Prevent Transplant Rejection

Mice (obtained from Jackson Laboratory) at 8 to 12 weeks of age wereanesthetized with Acepromazin maleate (Fermenta Animal Health Co.,Kansas City, Mo.). Prior to skin grafting, non-thymectomized recipientC57BL/6 mice (H-2^(b)) were injected intraperitoneally with 500 ug ofTAB4 or isotype control antibodies seven days before skin transplantsurgery. Seven days later, a lateral flank of skin from fully allogeneicmismatched BALB/cj mice (H-2^(d)) was grafted on the lateral flank ofthe antibody pre-treated C57BL/6 mice. Seven days post transplantation,the mice were again injected with 500 ug of TAB4 or isotype controlantibody. The mice were monitored every day after graft transplantation.The grafts were considered rejected when 50% donor skin was necrotic.The percent of graft survival is shown in FIG. 7 (n=8). The data showthat TAB4 antibody treatments prolonged the survival of the allogeneicskin grafts.

Example 9 Identification of TAIP as PSGL-1

P-selectin glycoprotein ligand-1 (PSGL-1), also named CD162, is the mainP-selectin ligand expressed on leukocytes, including T cells (Sako etal. (1993) Cell 75:1179; Vachino et al. (1995) J. Biol. Chem. 270:21966;Veldman et al. (1995) J. Biol. Chem. 270:16470). Biochemicalcharacteristics of TAIP, such as its molecular weight and its tendencyfor dimerization suggested the possibility that TAB4 may be analogous toPSGL-1. To investigate the relationship between these two antigens, thefollowing were tested: 1) whether the antigen precipitated by TAB4 canbe recognized by a commercially-available anti-PSGL1 antibody; and 2)whether an anti-PSGL1 antibody can deplete TAB4 from the cell lysate.

RL♂1 T cells were lysed at a density of 1.0×10⁸ cells/ml in lysis buffer(1% Triton X-100, 20 mM Tris-HCl, pH 8.0, 160 mM NaCl, 1 mM CaCl₂)containing complete protease inhibitor cocktail for 1 hour, andinsoluble material was pelleted at 10,000×g for 10 minutes. These andall subsequent steps were performed at 4° C. The lysate corresponding to5.0×10⁷ cells was incubated with 20 ul of protein G-Sepharose preloadedwith 10 ug of anti-PSGL-1 mAb (clone 2PH1, PHARMINGEN™, San Diego,Calif.), anti-TAIP mAb, TAB4, or IgG from normal hamster serum. Afterincubation for 4 hours at 4° C, the beads were washed five times withwashing buffer (0.05% Triton X-100, 50 mM Tris-HCl, pH 8.5, 400 mM NaCl,1 mM CaCl₂, 1 mg/ml ovalbumin), and twice with a similar washing buffer,containing 250 mM instead of 400 mM NaCl. Bound proteins were elutedwith 40 ul of 1×SDS sample buffer. Eluted proteins were separated by 6%SDS-PAGE and transferred to a nitrocellulose membrane. The membraneswere immunoblotted with anti-PSGL-1 mAb, and revealed byperoxidase-conjugated goat anti-rat IgG (H+L) followed bychemiluminescence (RENAISSANCE®, NEN™).

Surface biotinylated RL♂1 T cells were lysed at a density of 1.0×10⁸cells/ml in lysis buffer. The cell extract was incubated with 20 ug ofantibody bound to 40 ul of protein G-Sepharose at 4° C. overnight.Depletions were done with anti-PSGL-1 mAb (2PH1) or control rat IgG,with TAB4 or control normal hamster serum. Depleted lysates were furthersubjected to immunoprecipitation with TAB4 or anti-PSGL-1 mAb,respectively. Immunoprecipitates were separated on 6% SDS-polyacrylamidegel and detected by fluorography. As shown in FIG. 6, anti-PSGL-1antibody can deplete TAIP protein from T cell lysates. In addition,proteins immunoprecipitated with anti-TAIP antibody (TAB4) can berecognized by anti-PSGL-1 antibody by western analysis.

Example 10 Induction of Apoptosis in Human T Cells by an Anti-PSGL-1Antibody

To determine the role played by PSGL-1 in the apoptosis of human Tcells, time-course experiments were carried out to investigate whenactivated human T cells acquire sensitivity toward PSGL-1-mediatedapoptotic signals. Human T cells were stimulated with phytohemagglutinin(PHA) mitogen and further expanded in IL-2-containing medium. ActivatedT cells were harvested and then challenged with anti-PSGL-1 in thepresence of IL-2 and cross-linking antibodies.

Human peripheral blood was taken from healthy adults, heparinized, andenriched for peripheral blood mononuclear cells (PBMC) based ondifferential density using FICOLL-PLAQUE™ PLUS (Pharmacia Biotech). ThePBMC were activated with 1% PHA (Life Technologies, GibcoBRL) for 48hours and subsequently maintained in recombinant human IL-2 (5 ng/ml)through the assay period. To assess the apoptosis-inducing ability ananti-human PSGL-1 antibody, the activated cells were treated with: (1) 1ug/ml of the anti-PSGL-1 antibody clone KPL-1 (BD PHARMINGEN™) pluscross-linker rabbit anti-mouse Ig (0.5 ug/ml) (Jackson ImmunoResearchLaboratories); (2) isotype control purified mouse Ig plus cross-linkerrabbit anti-mouse Ig; or (3) cross-linker rabbit anti-mouse Ig alone.After six hours of treatment, the percentage of early apoptotic cellswas determined by FACS, staining with anti-Annexin V ( BD PHARMINGEN™)and PI (SIGMA®).

As shown in FIG. 8, signaling triggered by PSGL-1 using an anti-PSGL-1antibody plus the crosslinker triggered significant level of apoptosisin PHA-activated human PBMC (mainly T cells). The percentage ofapoptotic cells increased from 8.5% on day 3 to 24% on day 8 inanti-PSGL1 treated cultures. Neither isotype-matched control, nor thecross-linking antibodies alone, had any effect on these cells.

Example 11 Use of Anti-PSGL-1 Agonist Antibody to Treat AutoimmuneDisease

Non-obese diabetic (NOD) mice, a well-accepted autoimmune diabetesanimal, were bred under standard conditions. Spontaneous diabetesdeveloped in the NOD mice at the age of about 20 weeks. In theexperimental group, the mice received three doses of anti-PSGL-1antibody (TAB4) intraperitoneally at 300 μg per mouse at age of 14, 15and 17 weeks. Two additional injections with the same dose were given atthe ages of 24 and 26 weeks. The control group was given hamster Ig atthe same dose. Mice were monitored for glucosuria by Medi-Test Glucosestrips (Macherey-Nagel, Germany) twice every week after the age of 15weeks. Non-fasting urine glucose levels over 300 mg/dl for twoconsecutive measurements were considered diabetic.

As shown in FIG. 9, TAB4 (anti-PSGL-1) antibody treatment yieldedsignificant protection as compared with control antibody treatment. Thusan anti-PSGL-1 antibody treatment can dampen the activity of autoimmuneT cells and delay the onset of type I-diabetes.

Other Embodiments

It is to be understood that, while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention. Other aspects, advantages, and modifications of the inventionare within the scope of the claims set forth below.

1. A method of delaying or reducing a T cell-mediated immune response inan individual, the method comprising: selecting an anti-PSGL-1 antibodybased on its ability both to bind specifically to P-SelectinGlycoprotein Ligand-1 (PSGL-1) on the surface of an activated T cell andto induce apoptosis of the activated T cell; and administering to anindividual diagnosed as having a condition characterized by an excessiveor unwanted T cell-mediated immune response a composition comprising aneffective amount of the selected apoptosis-inducing anti-PSGL-1 antibodyor an antigen-binding fragment thereof to delay or reduce the Tcell-mediated immune response in the individual.
 2. The method of claim1, wherein the antibody is a monoclonal antibody.
 3. The method of anyone of claims 1 and 2, further comprising administering to theindividual an agent that binds to the antibody or antigen-bindingfragment thereof and induces cross-linking of a plurality of PSGL-1antigens on the surface of the T cell.
 4. The method of claim 1, whereinthe method comprises inducing cross-linking of a plurality of PSGL-1antigens on the surface of the T cell, wherein the cross-linking inducesa signal transduction pathway that results in death of the T cell. 5.The method of claim 1, wherein the condition characterized by anexcessive or unwanted T cell-mediated immune response is an autoimmunedisease.
 6. The method of claim 1, wherein the condition characterizedby an excessive or unwanted T cell-mediated immune response is rejectionof allogeneic or xenogeneic transplant.
 7. The method of claim 1,wherein the condition characterized by an excessive or unwanted Tcell-mediated immune response is an allergic disease.
 8. The method ofclaim 1, wherein the condition characterized by an excessive or unwantedT cell-mediated immune response is a T cell cancer.
 9. The method ofclaim 1, wherein the T cell is an activated T cell.
 10. The method ofclaim 1, wherein the T cell is a CD4⁺T cell.
 11. The method of claim 1,wherein the T cell is a CD8⁺T cell.
 12. The method of claim 1, furthercomprising: detecting a number of T cells in a first biological sampletaken from the individual before the administering; and comparing thenumber of T cells detected in the first biological sample with a numberof T cells in a second biological sample taken from the individual afterthe administering.
 13. The method of claim 1, further comprising:detecting a biological activity of T cells in a first biological sampletaken from the individual before the administering; and comparing thebiological activity of T cells detected in the first biological samplewith a corresponding biological activity of T cells in a secondbiological sample taken from the individual after the administering. 14.The method of claim 1, wherein the administering results in depletion ofat least 20% of peripheral blood CD3⁺cells in the individual.
 15. Amethod of inducing death of a T cell or natural killer (NK) cell, themethod comprising: selecting an anti-PSGL-1 antibody based on itsability both to bind specifically to P-Selectin Glycoprotein Ligand-1(PSGL-1) on the surface of an activated T cell or NK cell and to induceapoptosis of the activated T cell or NK cell; providing a T cell or NKcell expressing PSGL-1 on its cell surface; and contacting the T cell orNK cell with an effective amount of the selected apoptosis-inducingantibody or an antigen-binding fragment thereof to induce apoptosis ofthe T cell or NK cell.
 16. The method of claim 15, wherein the antibodyis a monoclonal antibody.
 17. The method of any one of claims 15 and 16,further comprising contacting the T cell or NK cell with an agent thatbinds to the antibody or antigen-binding fragment thereof and inducescross-linking of a plurality of PSGL-1 antigens on the surface of the Tcell or NK cell.
 18. The method of claim 15, wherein the methodcomprises inducing cross-linking of a plurality of PSGL-1 antigens onthe surface of the T cell or NK cell, wherein the cross-linking inducesa signal transduction pathway that results in death of the T cell or NKcell.
 19. The method of claim 15, wherein the T cell or NK cell is anactivated T cell.
 20. The method of claim 15, wherein the T cell or NKcell is a CD4⁺T cell.
 21. The method of claim 15, wherein the T cell orNK cell is a CD8⁺T cell.
 22. The method of claim 15, wherein the T cellor NK cell is an NK cell.
 23. A method of delaying or reducing a Tcell-mediated immune response in an individual, the method comprising:selecting an anti-PSGL-1 antibody based on its ability both to bindspecifically to P-Selectin Glycoprotein Ligand-1 (PSGL-1) on the surfaceof an activated T cell and to induce apoptosis of the activated T cell;and administering to an individual diagnosed as having or as being atrisk of acquiring a condition characterized by an excessive or unwantedT cell-mediated immune response a composition comprising an effectiveamount of the selected apoptosis-inducing anti-PSGL-1 antibody or anantigen-binding fragment thereof to delay or reduce the T cell-mediatedimmune response in the individual, and wherein the conditioncharacterized by an excessive or unwanted T cell-mediated immuneresponse is an autoimmune disease, rejection of allogeneic or xenogeneictransplant, an allergic disease or a T cell cancer.